ETV2/ER71 Regulates the Generation of FLK1(+) Cells from Mouse

ETV2/ER71 regulates the generation of FLK1(+) cells from mouse embryonic stem cells through miR-126-MAPK signaling JY Kim, Emory University DH Lee, Emory University JK Kim, Emory University HS Choi, Emory University Bhakti Dwivedi, Emory University M Rupji, Emory University Jeanne Kowalski, Emory University H Song, Emory University JY Chang, Emory University Changwon Park, Emory University

Journal Title: Stem Cell Research and Therapy Volume: Volume 10, Number 1 Publisher: BMC | 2019-11-19, Pages 328-328 Type of Work: Article | Final Publisher PDF Publisher DOI: 10.1186/s13287-019-1466-8 Permanent URL: https://pid.emory.edu/ark:/25593/vh4ms

Final published version: http://dx.doi.org/10.1186/s13287-019-1466-8 Copyright information: © 2019 The Author(s). This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/). Accessed September 26, 2021 9:42 PM EDT Kim et al. Stem Cell Research & Therapy (2019) 10:328 https://doi.org/10.1186/s13287-019-1466-8

SHORT REPORT Open Access ETV2/ER71 regulates the generation of FLK1+ cells from mouse embryonic stem cells through miR-126-MAPK signaling Ju Young Kim1,2,3, Dong Hun Lee1,2, Joo Kyung Kim1,2, Hong Seo Choi1, Bhakti Dwivedi5, Manali Rupji5, Jeanne Kowalski5,6,7, Stefan J. Green8, Heesang Song1,9, Won Jong Park1, Ji Young Chang1, Tae Min Kim10 and Changwon Park1,2,3,4*

Abstract Previous studies including ours have demonstrated a critical function of the transcription factor ETV2 (ets variant 2; also known as ER71) in determining the fate of cardiovascular lineage development. However, the underlying mechanisms of ETV2 function remain largely unknown. In this study, we demonstrated the novel function of the miR (micro RNA)-126-MAPK (mitogen-activated protein kinase) pathway in ETV2-mediated FLK1 (fetal liver kinase 1; also known as VEGFR2)+ cell generation from the mouse embryonic stem cells (mESCs). By performing a series of experiments including miRNA sequencing and ChIP (chromatin immunoprecipitation)-PCR, we found that miR-126 is directly induced by ETV2. Further, we identified that miR-126 can positively regulate the generation of FLK1+ cells by activating the MAPK pathway through targeting SPRED1 (sprouty-related EVH1 domain containing 1). Further, we showed evidence that JUN/FOS activate the enhancer region of FLK1 through AP1 (activator protein 1) binding sequences. Our findings provide insight into the novel molecular mechanisms of ETV2 function in regulating cardiovascular lineage development from mESCs. Keywords: ETV2/ER71, FLK1+ cells, miR-126, EGFL7, Embryonic stem cells

Introduction compartments [5, 6]. We also showed that ETV2 can dir- The ETS (E26 transformation-specific or E-twenty-six ectly bind the promoters/enhancers of endothelial and specific sequence) transcription factor family members hematopoietic genes including Flk1 (fetal liver kinase 1; play key roles in diverse biological events including cell also known as VEGFR2) [5–7]. Together with the findings survival, cancer, vascular-angiogenesis and hematopoiesis that ETV2 can induce de novo generation of FLK1+ cells, mainly through the interaction between their conserved the multipotent progenitor for blood, endothelial and car- ETS DNA binding domain and the consensus sequence diac lineages from mouse embryonic stem cells (mESCs) (5′-GGAA/T-3′) present in the regulatory elements of [5], these results strongly suggest the critical function of their target genes [1, 2]. ETV2 (ets variant 2; also known ETV2 for the establishment of the cardiovascular system. as ER71), a member of the ETS transcription factor family, Reports from other groups further support the importance has been reported as an indispensable factor in establish- of ETV2 in this process [8–11]. Regarding the regulatory ing the cardiovascular system [3, 4]. We previously dem- mechanisms of ETV2 functions, several studies examining onstrated that deficiency in Etv2 led to embryonic lethality the ETV2 binding proteins have been reported. For ex- due to a complete lack of both vascular and hematopoietic ample, it was shown that the interaction between ETV2 and FOXC2 (forkhead box protein C2) plays an important role in regulating several key genes of the endothelial and * Correspondence: [email protected] 1Department of Pediatrics, Emory University School of Medicine, 2015 hematopoietic lineages [12, 13]. Also, our recent study re- Uppergate Dr. Atlanta, Atlanta, GA 30322, USA vealed the functional significance of the ETV2-OVOL2 2 Children’s Heart Research & Outcomes (HeRO) Center, Children’s Healthcare (ovo-like zinc finger 2) interaction in generating FLK1+ of Atlanta & Emory University, Atlanta, GA, USA Full list of author information is available at the end of the article cells and its further differentiation into the hematopoietic

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kim et al. Stem Cell Research & Therapy (2019) 10:328 Page 2 of 9

and endothelial cells [14]. However, the detailed molecular ETV2 ESCs upon Dox treatment (Figs. 1e and 2a). Next, insight into the ETV2 function remains largely unknown. we examined whether ETV2 can directly activate the To better understand the machinery of ETV2 that promoter of Egfl7, thus inducing the expression of miR- regulates the FLK1+ cell generation from mESCs, we 126. From the literature search and our own investiga- profiled miRNAs (micro RNAs) that are differentially tion, we found the highly conserved upstream region of regulated by ETV2 and found miR-126 as one of the the transcription start site in mouse and human EGFL7 direct downstream players of ETV2. We subsequently that contains two conserved potential ETS binding sites investigated the molecular mechanism of the miR-126/ (Fig. 2b). By performing the promoter assay, we revealed MAPK (mitogen-activated protein kinase) pathway in that the overexpression of Etv2 significantly increased ETV2-mediated FLK1+ cell generation. the activity of the Egfl7 promoter. However, the Egfl7 promoter construct with mutations on one putative ETS Materials and methods site failed to respond to ETV2 (Fig. 2c). The results were Complete materials and methods are presented in further corroborated by chromatin immunoprecipitation Additional file 1: Supplemental materials and methods. (ChIP)-PCR assay, confirming in vivo occupancy of ETV2 in Egfl7 promoter (Fig. 2d). Taken together, we Results conclude that the expression of Egfl7 and thus miR-126 Analysis of ETV2-mediated miRNAs is directly regulated by ETV2 in differentiating mESCs. To gain a novel insight into the molecular mechanisms of ETV2 function in FLK1+ cell generation, we performed The miR-126/MAPK pathway plays an important role for miRNA profiling analysis. FLK1+ cells from doxycycline ETV2-induced FLK1+ cell generation (Dox)-inducible ETV2 in mESC (herein, iFLAG-ETV2 MiR-126 (especially, miR-126-3p) can regulate the ESCs) [14]atday3.5ofdifferentiation±DoxwereFACS MAPK pathway through the suppression of the SPRED1 (fluorescence-activated cell sorting)-sorted and subjected to expression, a negative regulator of the MAPK pathway miRNA sequencing (Fig. 1a). The miRNAs with ≥ 1.5 fold [15, 19]. Together with the report that treatment of change and a false discovery rate (FDR) ≤ 0.05 were consid- bFGF (basic fibroblast growth factor), an agonist of FGF ered to be significantly differentially expressed, resulting in receptor-mediated signaling including MAPK [20], in- a total of 67 miRNAs of interest that were subsequently creases the generation of the FLK1+ cells from mESCs subjected to unsupervised hierarchical clustering (Fig. 1b, [21], we hypothesized that ETV2 induces FLK1+ cells c). GO (gene ontology) term analysis indicated that the partly through the miR-126-MAPK pathway by sup- ETV2-mediated miRNAs could be involved in diverse bio- pressing the expression of SPRED1. As shown in Fig. 3a logical events with embryo development, cell differentiation and b, while SPRED1 (sprouty related EVH1 domain and anatomical structure development being top ranked containing 1) was significantly reduced in response to (Fig. 1d). Signaling pathways such as MAPK, RAP1 (ras-as- ETV2, augmented phospho-ERK1/2 (extracellular signal- sociated protein 1) and WNT (wingless-related integration regulated kinases 1/2) level was evident upon the ETV2 site) were identified as the major regulatory network of the overexpression in differentiating mESCs. Interestingly, − − miRNAs, all of which are critical for cardiovascular devel- Etv2 / ESCs showed an increased level of SPRED1 ex- opment (Additional file 2: Tables S1 and S2). Some of the pression with concomitant decrease of phosphorylated differentially expressed miRNAs were validated by qRT- ERK1/2. Further, we found that ETV2-induced FLK1+ PCR (Fig. 1e and Additional file 3:FigureS1). cell generation was significantly reduced in the presence Among the differentially regulated miRNAs, miRNA- of U0126, a MAPK inhibitor, compared to the control 126 drew our attention as an important player of ETV2- (Fig. 3c). To corroborate the findings, we transfected mediated FLK1+ cell generation due to its function in Dox-inducible MAP2K1-8E, a catalytically inactive form vascular development and hematopoiesis [15–17]. Inde- of MAP2K1 [22] into iFLAG-ETV2 mESCs in which pendently, our previous reports showed that Egfl7 (egf- both ETV2 and MAP2K1-8E are co-overexpressed upon like domain multiple 7) [18], the host gene of miR-126, Dox treatment. In agreement with the inhibitor treat- is one of the most upregulated genes upon ETV2 over- ment (Fig. 3c), the co-expression of MAPK2K1-8E and expression [6, 7]. Accordingly, we sought to determine ETV2 led to a decreased generation of FLK1+ cells, com- the functional consequence of miR-126 in regulating pared to the group in which ETV2 only is overexpressed ETV2-mediated FLK1+ cell generation. (Fig. 3d). Additionally, we went on to show that overexpression of SPRED1 was able to inhibit generation of ETV2 directly activates the expression of miR-126 through FLK1+ cells upon the overexpression of ETV2 (Fig. 3d). Egfl7 promoter These results clearly suggest that the SPRED1/MAPK First, we found a significant increment of the expression pathway plays an important role for ETV2-induced of both miR-126 and Egfl7 in differentiating iFLAG- FLK1+ cell generation from mESCs. Kim et al. Stem Cell Research & Therapy (2019) 10:328 Page 3 of 9

Fig. 1 Analysis of ETV2-regualted miRNA expression in FLK1+ cells. a Schematic diagram of miRNA sequencing experiment. Doxycycline-inducible (iFLAG-ETV2) mESCs were differentiated, treated with ± Doxycycline (Dox) at day 1 and sorted for FLK1+ cells at day 3.5. RNAs from the sorted cells were subjected to miRNA sequencing and analysis. b Volcano plot showing the log2 fold change between +Dox versus −Dox against the −log10 FDR-adjusted p value for each miRNA. miRNAs (FDR ≤ 0.05) with fold change of ≥ 1.5 (in red; upregulated) and ≤−1.5 (in green; downregulated) were highlighted and selected. c Heatmap of the selected miRNAs in response to overexpression of ETV2. miRNAs upregulated and downregulated in +DOX were indicated with gray and black bars, respectively. d Gene Ontology (GO) categories of selected miRNAs by DIANA miRpath analysis. Bars indicate the significance level of miRNA target genes and interactions. e Differentiated iFLAG-ETV2 mESCs at day 3.5 were subjected to qRT-PCR analysis. n = 3, ***p < 0.001

Direct activation of FLK1 gene expression by the MAPK mechanistic link between ETV2 and FLK1 gene ex- pathway pression via the MAPK-AP1 pathway. Therefore, we To get a detailed insight into how the MAPK pathway first performed a luciferase-based promoter assay and activated by ETV2 regulates the generation of FLK1+ revealed that the activity of the Flk1-promoter/enhan- cells, we examined the regulatory elements such as pro- cer (p/e) was increased in response to ETV2 or c- moter and enhancer of Flk1 gene [23] and found several JUN/FOS, a downstream of the MAPK pathway potential activator protein 1 (AP1) binding sequences in (Fig. 4b, c). In contrast, both MKAP2K1-8E and the Flk1 enhancer which is critical for controlling the en- JUN dominant negative mutant, c-JUN DN [26], dogenous expression of Flk1 (Fig. 4a). AP1 (activator inhibitedETV2functioninactivatingFlk1-p/e protein 1), a heterodimeric transcriptional complex con- (Fig. 4b). Further, we showed that mutations on the sisting of JUN and FOS proteins, conveys the important putative AP-1 binding site in the Flk1-p/e led to a functions for multiple biological processes such as differ- significantly reduced luciferase activity induced by c- entiation, proliferation and apoptosis [24]. Given the JUN/FOS (Fig. 4c). Collectively, these results suggest findings that AP1 complex acts as a downstream target that the MAPK pathway can activate the expression of the MAPK pathway [25], these suggest an important of Flk1 gene partly through c-JUN/FOS. Kim et al. Stem Cell Research & Therapy (2019) 10:328 Page 4 of 9

Fig. 2 ETV2 upregulates miR126 expression through direct binding on Egfl7 promoter. a Expression analysis. iFLAG-ETV2 mESCs were differentiated for 3.5 days ± Dox treatment and were subjected to gene expression analysis. n =3,*p < 0.05. b Schematic diagram of Egfl7 promoter-luciferase plasmid. Two potential ETS binding elements were marked as diamonds, and their mutant sequences were shown in the bottom. c HEK/293T cells were transiently co-transfected with pCMV-Etv2 and pGL3-luciferase constructs carrying wild type (Wt), or mutants of Egfl7 promoter. Firefly luciferase activity was normalized by Renilla luciferase activity. n = 3. ***p < 0.001. d iFLAG-ETV2 mESCs were differentiated in the presence of Dox for 3.5 days and then subjected to ChIP-PCR assay. Rabbit anti-FLAG or IgG antibody was used for the immunoprecipitation. n =3,**p < 0.01

Discussion CXCR4 (C-X-C chemokine receptor type 4), a receptor In our study, several layers of novel findings on ETV2 of SDF1 (stromal cell-derived factor 1), whose signaling function were made. First, we reported genome-wide is important during heart development and promotes miRNA profiles in FLK1+ cells generated in response to cardiogenesis from pluripotent stem cells [28–31]. An- ETV2, providing a new research resource. Second, we other downregulated miRNA by ETV2, miR-106, has demonstrated the functional significance of the miR- been reported as a critical player for cardiac develop- 126/MAPK pathway in ETV2-mediated FLK1+ cell gen- ment as evidenced by in utero lethality displaying severe eration. Third, we also showed a direct activation of Flk1 cardiac defects in compound knockout of mouse miR- enhancer by the JUN/FOS complex. Overall, our results 106~25 and miR-17~92 cluster [32]. Thus, determining reveal that the miR-126/MAPK pathway constitutes a biological consequences of the links between ETV2 and novel mechanism responsible for ETV2-mediated Flk1 ETV2-dependent miRNAs for cardiovascular lineage de- expression (Fig. 4d). velopment would be an interesting topic for further Our miRNA profiling results suggest that ETV2 regu- studies. lates development of FLK1+ cells, hematopoietic and Studies have shown that miR-126 plays important endothelial cell lineages as well as cardiomyocytes functions in cardiovascular development and angiogen- through miRNAs. For example, miR-10b, one of the esis [15–17]. In this study, we provided evidence of the downregulated miRNAs by ETV2, is a critical regulator unknown function of miR-126 in generating the first for vessel development through targeting FLT-1 (fms-re- emerging FLK1+ cells. Our results showed that ETV2 via lated tyrosine kinase 1), which can inhibit VEGF (vascu- a direct binding on the Egfl7 promoter can induce the lar endothelial growth factor)-FLK1 signaling [27]. In expression of miR-126. In the subsequent studies, inhib- contrast, miR-146a was upregulated in response to ition of the miR-126/MAPK pathway leads to impair- ETV2 expression. Interestingly, miR-146 can target ment of ETV2-mediated FLK1+ cell generation. Thus, Kim et al. Stem Cell Research & Therapy (2019) 10:328 Page 5 of 9

Fig. 3 (See legend on next page.) Kim et al. Stem Cell Research & Therapy (2019) 10:328 Page 6 of 9

(See figure on previous page.) Fig. 3 ETV2 increases FLK1+ cells through the miR-126/MAPK pathway. A, B iFLAG-ETV2 mESCs were differentiated with ± Dox for 3.5 days and subjected to western blot analysis for SPRED1 (A) and phosphorylated ERK1/2 (B). β-ACTIN and ERK1/2 were used as loading controls. (A’, B’) The relative protein expression of SPRED1 and p-ERK1/2 was normalized against β-ACTIN and total ERK1/2, respectively. Differentiated Etv2−/− mESCs were also included for the analysis. n =3,*p < 0.05, **p < 0.01. (C) iFLAG-ETV2 mESCs were differentiated for 4 days in serum-free (upper) or for 3.5 days in serum (lower) conditions in the presence or absence of Dox. The resulting cells were analyzed for FLK1 expression by flow cytometry. U0126 (5 μM) or U0124 (5 μM) was treated during the differentiation. C’ Quantification. n =3,*p < 0.05. D Differentiated iFLAG-ETV2, iFLAG-ETV2- MAP2K1-8E (iFLAG-ETV2-M) or iFLAG-ETV2-SPRED1 (iFLAG-ETV2-S) mESC ± Dox were analyzed for FLK1 expression by flow cytometry. D’ Quantification. n =3,*p < 0.05

Fig. 4 ETV2 activates FLK1 expression through AP-1 binding sites in Flk1 enhancer region. a Schematic diagram of Flk1-promoter-enhancer (p/e)- luciferase plasmid. Three potential AP-1 binding elements were marked as diamonds. b, c Luciferase-based promoter assay. b pGL3-Flk1 p/e was transiently co-transfected with pCMV-Etv2, pMCL-MAP2K1-8E, pCMV-c-Jun ND (dominant negative form of c-Jun) into HEK/293T cells. n =3,**p < 0.01, ***p < 0.001. c pGL3-luciferase construct carrying wild type (Wt), putative AP-1 binding site mutants (Mt #1, Mt #2, Mt #3) or enhancer deletion mutant (del) of Flk1 p/e was co-transfected with pCMV-c-Jun and c-Fos into HEK/293T cells. Firefly luciferase activity was normalized by Renilla luciferase activity. n = 3, ***p < 0.001. d A working model for ETV2-miR126/MAPK in regulating FLK1 expression. In addition to the direct binding of ETV2 on the ETS binding sites in FLK1 gene, ETV2 activates miR-126, which can target SPRED1, thereby activating the MAPK pathway. JUN/FOS, a downstream signaling complex of the MAPK pathway, subsequently activates gene transcription of FLK1 via the AP1 binding sites Kim et al. Stem Cell Research & Therapy (2019) 10:328 Page 7 of 9

our results suggest a novel post-transcriptional regula- expression of Flk1, which could provide an in-depth and tory mechanism of ETV2 in generating FLK1+ cell. Al- novel insight into molecular mechanisms behind the though EGFL7 is co-transcribed with miR-126 upon ETV2-FLK1 axis. ETV2 expression, we rule out the potential contribution In conclusion, we reported miRNA profiles regulated of EGFL7 to inducing FLK1+ cell generation due to the by ETV2 in generating FLK1+ cells from mESCs. Fur- dispensable function of EGFL7 in early stages of em- ther, we showed that ETV2 can regulate the expression bryogenesis and embryonic vasculature formation as of Flk1 through the miR126/MAPK pathway. These demonstrated by knockout studies in mice and knock- findings could provide a novel insight into the mecha- down experiments in differentiating ESCs [18, 33]. nisms of how ETV2 regulates the development of the Taken together, it is evident that miR-126 acting down- cardiovascular system. Studying the functions of other stream of ETV2 plays important roles in the establish- miRNAs identified in this research would be important ment of the cardiovascular system. for further deciphering the ETV2-miRNA regulatory The molecular insights into Flk1 gene expression (i.e., mechanisms in FLK1+ cell and cardiovascular lineage determining its upstream regulators) has not been an ac- generation. Since embryonic events can often play a crit- tive research area, despite its critical function in ical role in adults, examination of our findings in patho- hematopoietic and vascular system. Regarding this, we physiological angiogenesis would be warranted. have demonstrated that ETV2 directly binds ETS binding elements present on both promoter and enhancer of Supplementary information Flk1 and activates its expression [5]. The subsequent Supplementary information accompanies this paper at https://doi.org/10. 1186/s13287-019-1466-8. ChIP sequencing analysis demonstrated that a wide range of genes critical for endothelial cell and blood cells Additional file 1. Supplemental materials and methods are direct downstream targets of ETV2 [7]. In this Additional file 2: Table S1. Gene Ontology (GO) categories for genes current study, we revealed an important function of targeted by significant differentially expressed miRNA’s. Table S2. KEGG MAPK activity in regulating Flk1 gene expression. Over- categories for genes targeted by significant differentially expressed ’ expression of ETV2 activates the MAPK activity, while miRNA s. Table S3. Primer sequences. inhibition of the MAPK pathway decreases ETV2- Additional file 3: Figure S1. Analysis on miR-126-5p in response to + ETV2. Differentiated iFLAG-ETV2 mESCs at day 3.5 were subjected to qRT- mediated FLK1 cell generation. Further, we showed PCR analysis. n=3, **p<0.01. that the MAPK pathway is able to directly upregulate the expression of Flk1 via a direct binding of JUN/FOS Abbreviations on Flk1 enhancer. Thus, we envision that ETV2 induces AP1: Activator protein 1; ChIP: Chromatin immunoprecipitation; c-JUN DN: JUN dominant negative mutant; CXCR4: C-X-C chemokine receptor type Flk1 gene expression in a bimodal manner, one by acti- 4; Dox: Doxycycline; EGFL7: Egf-like domain multiple 7; ERK1/2: Extracellular vating the miR-126/MAPK pathway and the other by its signal-regulated kinases 1/2; ETS: E26 transformation-specific; ETV2: Ets direct binding on Flk1 gene regulatory elements. In variant 2; FACS: Fluorescence-activated cell sorting; FDR: False discovery rate; FLK1: Fetal liver kinase 1; Flk1-p/e: Flk1-promoter/enhancer; FLT-1: Fms- mouse embryos, activated ERK1/2 is detected in the related tyrosine kinase 1; FOXC2: Forkhead box protein C2; GO: Gene blood islands, the first sites of blood and endothelial cell ontology; iFLAG-ETV2: Doxycycline-inducible FLAG-ETV2; MAPK: Mitogen- development and later in the dorsal aorta and intersomi- activated protein kinase; mESCs: Mouse embryonic stem cells; OVOL2: Ovo- like zinc finger 2; RAP1: Ras-associated protein 1; SDF1: Stromal cell-derived tic vessels [34]. Further, Erk2-deficient mouse embryos factor 1; SPRED1: Sprouty-related EVH1 domain containing 1; VEGF: Vascular are incompatible with proper mesoderm formation [35, endothelial growth factor; WNT: Wingless-related integration site 36]. Sprouty/Spred proteins, the negative effectors for Ras/MAPK pathway [19], have inhibitory functions in Acknowledgements We would like to thank Dr. Seyoung Oh for initial technical support. We also generating mesoderm and blood as well as vessel devel- would like to thank Emory’s Pediatrics/Winship Flow Cytometry Core. opment [37–40], further suggesting important functions of the MAPK pathway in emergence of mesoderm with Authors’ contributions JYK, DHL, JKK, HSC, HS, WJP, JYC, and TMK contributed to the collection and hematopoietic and endothelial potential. Considering assembly of data. JYK and DHL contributed to the design, data analysis, that the enhanced protein stability of ETV2 by OVOL2 interpretation, and manuscript writing. BD, MR, JK, and SJG contributed to is thought to be one mechanism for ETV2-mediated the data analysis. CP contributed to the conception, design, collection and assembly of data, financial support, data interpretation, and manuscript Flk1 gene transcription [14], it would be interesting to writing. JYK and CP contributed to the final approval of manuscript. All see if the interaction between ETV2 and OVOL2 can authors read and approved the final manuscript. positively regulate the miR-126/MAPK pathway in this Funding process. In addition, the findings that ETV2 interacts Research reported in this publication was supported in part by the with OVOL2 [14] or FOXC2 [12] and that ETV2 and Biostatistics and Bioinformatics Shared resource of Winship Cancer Institute JUN/FOS acts on Flk1 promoter and enhancer suggest a of Emory University and NIH/NCI under award number P30CA13829. This work was also supported by Basic Science Research Program through the hypothesis that ETV2 can form a transcriptional com- National Research Foundation of Korea (NRF) funded by the Ministry of plex with OVOL2, FOXC2, and/or AP1 to regulate the Education (2018R1D1A1A02085481) (T.M.K) and the March of Dimes Kim et al. Stem Cell Research & Therapy (2019) 10:328 Page 8 of 9

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